JP2022036947A - Self-organized peptide composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a peptide composition (for example, the specific composition of a self-organized peptide agent) having a specific attribute (for example, peptide identity, peptide concentration, a pH, ionic intensity) showing especially useful material properties and the like, and a technique for selecting and/or preparing a specific peptide composition useful in a specific context.

SOLUTION: A liquid peptide composition including peptide (for example: IEIK13, RADA16, KLD12 peptide) having an amino acid length within approximately 6-20 pieces and an alternate amino acid sequence of hydrophobic amino acid and hydrophilic amino acid forms gel within approximately 30 seconds when exposed/maintained to a pH of approximately 2.5-4.0 and/or an ionic intensity of approximately 0.0001M-1.5M.

SELECTED DRAWING: None

COPYRIGHT: (C)2022,JPO&INPIT

Description

[Cross-reference of related applications]
This application claims the benefit of US Provisional Patent Application No. 61 / 950,529, filed March 10, 2014, under US Code Vol. 35, Section 119 (e). , Which is incorporated herein by reference in its entirety.

[Array list]
This application is based on ASCII. Refer to the sequence list submitted electronically as a txt file. The txt file was created on March 9, 2015 and is 1 kb in size.

Peptide agents capable of self-assembling into gel structures have a wide variety of uses in therapeutic and research contexts. One such peptide agent, eg, a synthetic 16-amino acid polypeptide having a repeating sequence of arginine, alanine, and aspartic acid (ie, RADARADARADARADA [SEQ ID NO: 1], also known as "RADA16". Are marketed from 3-D Matrix Medical Technology under the trade names PuraStat®, PuraMatrix®, and PuraMatrix GMP®, cell culture, drug transport, promoted cartilage and bone growth. , And in a wide range of experimental and clinical applications involving regeneration of CNS, soft tissue, and myocardium, as well as matrices, scaffolds, or one or more detectable agents, biologically active agents, cells, and / or. Its usefulness has been shown as a tedder that may be associated with cellular components.

The present disclosure provides, among other things, specific peptide compositions (specifically, specific compositions of self-assembling peptide agents) and techniques relating thereto. In some embodiments, such a composition may or may be a solution. In some embodiments, such a composition may or may be a gel. In some embodiments, such compositions may or may be solid (eg, dried / lyophilized) peptides.

For example, in the present disclosure, a particular peptide composition (ie, a peptide composition having a particular concentration, ion intensity, pH, viscosity and / or other properties) may have useful and / or surprising attributes (eg, gelation). Or have self-assembling kinetics [eg, rate of gelation and / or rate and reversibility of peptide self-assembling], rigidity [eg, as assessed by storage modulus], and / or other mechanical properties. Show that. In some embodiments, the present disclosure demonstrates the particular usefulness of a particular composition in a particular context (eg, a particular in vivo or in vitro application).

In particular, the present disclosure provides guidelines that allow the selection, design, and / or formulation of specific peptide compositions useful in a particular context or application.

The present disclosure discloses the extent to which specific cations and anions interact with self-assembling peptide agents, and how such interactions alter specific substance (eg, rheology) properties of the peptide composition. Establish what can be done (eg, increase mechanical rigidity and / or viscosity). Furthermore, the present disclosure describes how such interactions are gelled kinetics, restoration of the gelled state (eg, after exposure to deformation (eg, mechanical perturbation or other disintegration), among other things). For example, establish whether it can affect the timing and / or degree of gelation and / or restoration of gel properties.

The tests described herein identify the causes of various problems with certain existing self-assembling peptide techniques, and are particularly useful and / or necessary specific to the particular application of the peptide composition technique. Define attributes and / or characteristics.

In some embodiments, the peptide contained in the provided composition is a self-assembling peptide. In some embodiments, the peptide contained in the provided composition is an amphipathic peptide. In some embodiments, the peptides contained in the provided composition are at least continuous (eg, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16). , 17, 18, 19, 20 amino acids, etc.) have an amino acid sequence characterized by alternating hydrophilic and hydrophobic amino acids. In some embodiments, the peptide contained in the provided composition has an amino acid sequence comprising one or more repeating Arg-Ala-Asp-Ala (RADA). In some embodiments, the peptides contained in the provided composition have an amino acid sequence comprising or consisting of repeating units of the sequence Lys-Leu-Asp-Leu (KLDL). In some embodiments, the peptides contained in the provided composition have an amino acid sequence comprising or consisting of repeating units of the sequence Ile-Glu-Ile-Lys (IEIK). In some embodiments, the peptide can be IEIK13, KLD12, or RADA16. In some embodiments, the compositions of these peptides may have enhanced properties compared to suitable reference compositions with different (eg, lower) pH levels and / or ionic strength. ..

Peptide compositions at milder pH levels can have more rigid rheological properties, making them suitable for a wider range of applications. Changes in environmental pH above 4.0 can also have a beneficial effect on gelation kinetics with the peptide composition. In some embodiments, the pH increase can be a physiological pH that can occur when the peptide composition is placed in the body.

According to one or more embodiments, the rheological properties of certain peptide compositions, including but not limited to IEIK13, KLD12, and RADA16, can be enhanced by maintaining increased ionic strength. In some embodiments, the ionic strength may be lower than the critical ionic strength. In some embodiments, the peptide composition may be dissolved in water containing salts rather than pure water. In some embodiments, the ionic strength may be lower than their critical ionic strength.

In some embodiments, the increased ionic strength can have a favorable effect on the stiffness and / or gelling kinetics on the peptide composition, making them suitable for a wider range of applications. In some embodiments, the increased ionic strength can be the physiological ionic strength that can occur when the peptide composition is placed in the body.

According to one or more embodiments, the properties of certain peptide compositions, including but not limited to IEIK13, KLD12, and RADA16, are such that their pH level is about 3.5 or less, and at the same time their salt concentration. , Can be enhanced by keeping them below critical ionic strength levels (ie, no precipitation).

According to one or more embodiments, the self-assembling peptide (eg, IEIK13, KLD12, and RADA16) has various applications of appearance, pH level, ionic strength level, gelling kinetics, rheological properties, and peptide formulation. It can be characterized in terms of properties, including cell viability that is optimized for. IEIK13 and KLD12 can be characterized as peptide compositions similar to RADA16 in terms of basic gelling properties and other characteristics.

In some embodiments, the peptide may have an amino acid length in the range of about 6 to about 20 and an alternating amino acid sequence of hydrophobic and hydrophilic amino acids.

In some embodiments, the peptide composition can be a solution, a gel, or any combination thereof.

In some embodiments, the peptide composition may be at a concentration of at least 0.05%. In some embodiments, the peptide composition may be present at a concentration of less than 3%.

In some embodiments, the peptide composition may have a pH in the range of about 2.5 to about 4.0 or in the range of about 3.0 to about 4.0. In some embodiments, the pH of the peptide composition is sodium hydroxide or potassium hydroxide, calcium hydroxide, sodium carbonate, sodium acetate, sodium sulfide, DMEM (Dalbeco's Modified Eagle's Medium), and PBS (Phosphate Buffered Phosphate). It can be achieved by a solution selected from the group consisting of saline).

In some embodiments, the ionic strength of the peptide composition can be from about 0.0001M to about 1.5M. In some embodiments, the ionic strength of the peptide composition is by mixing common salts such as NaCl, KCl, MgCl2, CaCl2, CaSO4, DPBS (Dalvecolinic acid buffered saline, 10x). Can be adjusted. In some embodiments, the ionic intensity of the peptide composition can be adjusted by mixing common salts, where the one or more common salts can be one or more to form a cation. The salt consisting of one or more salts forming the anion and the salt forming the cation is selected from the group consisting of ammonium, calcium, iron, magnesium, potassium, pyridinium, quaternary ammonium, and sodium. The salt forming the anion is selected from the group consisting of acetate, carbonate, chloride, citrate, cyanide, floride, nitrate, nitrite, and phosphate.

In some embodiments, the peptide composition may have a viscosity in the range of about 1 to about 10000 Pa · S. In some embodiments, the peptide composition may have a storage modulus in the range of about 50 to about 2500 Pa.

In some embodiments, the method of selecting a peptide composition for application to a particular in vivo site comprises the storage modulus, viscosity, gelling time, time and / or degree of restoration, etc. for the peptide composition. A step of determining one or more parameters selected from the group, a step of comparing the determined parameters to specifications for various applications, a step of selecting a peptide composition in the light of comparison; and a selected peptide composition. It may include the step of administering the substance to the site.

In some specific embodiments, the disclosure may comprise, for example, peptides having amino acid lengths ranging from about 6 to about 20 and alternating hydrophobic and hydrophilic amino acid sequences. Provided are liquid peptide compositions that can be characterized in the following respects: (I) At room temperature, it has a viscosity in the range of about 1 Pa · s to about 500,000 Pa · s; (ii) with a frequency of 1 rad / sec and a vibration stress of 1 Pa, it is in the range of about 1 to about 5000 Pa. And / or (iii) exposed to pH in the range of about 2.5 to about 4.0 and / or ionic strength in the range of about 0.0001M to about 1.5M. When maintained, it forms a gel for about 0 to about 30 s. In some embodiments, such a composition is an aqueous composition.

Also, in some specific embodiments, the present disclosure provides a method of designing, selecting, or producing a peptide composition that is particularly suitable for use in a particular context. In some such embodiments, a particular context is or comprises an application to a particular in vivo site. In some embodiments, such provided method is selected from the group consisting of, for example: (i) storage modulus, viscosity, gelling time, shear thinning properties, peptide nanofiber reassembly time. Steps to determine one or more parameters and / or one or more other parameters described herein suitable for application to a particular in vivo site; and (ii) provided herein. According to the guidelines given, the steps of designing, selecting, and / or producing a peptide composition characterized by such parameters may be included.

Alternatively or in addition, in some specific embodiments, the present disclosure provides a method of selecting a particular peptide composition, eg, for administration to a particular in vivo site; exemplary thereof. Such methods are one or more parameters selected from the group consisting of (i) storage modulus, viscosity, gelling time, shear thinning properties, peptide nanofiber reassembly time, and / or peptide composition. Steps to determine one or more other parameters described herein with respect to an object; (ii) Determined that the determined one or more parameters are suitable for application to a particular in vivo site. It may include a step of comparing to a set of properties; (iii) selecting a peptide composition in the light of comparison; and (iv) administering to the site the selected peptide composition.

The objects and features of the present invention can be better understood with reference to the drawings described below and the claims.

Illustrative gel formation of peptide compositions in PBS buffer is shown. RADA16, IEIK13, and KLD12 were plated at different concentrations of 0.5%, 1.0%, 1.5%, 2.0% and 2.5%. RADA16, IEIK13, and KLD gelled at all concentrations. 2A and 2B show exemplary rheological properties of RADA16. FIG. 2A shows stress sweep tests performed at 1 Pa and 10 rad / s. FIG. 2B shows the measured storage modulus as a function of RADA16 concentration. The storage modulus of the RADA16 composition may have a linear correlation with their concentration. 3A and 3B show exemplary rheological properties of IEIK13. FIG. 3A shows stress sweep tests performed at 1 Pa and 10 rad / s. FIG. 3B shows the measured storage modulus as a function of the IEIK concentration. The storage modulus of the IEIK13 composition may have a linear correlation with their concentration. 4A and 4B show exemplary rheological properties of KLD12. FIG. 4A shows stress sweep tests performed at 1 Pa and 10 rad / s. FIG. 4B shows the measured storage modulus as a function of KLD12 concentration. The storage modulus of the KLD12 composition may have a linear correlation with their concentration. 5A and 5B are bar graphs showing the effect of DMEM (Dulvecco's Modified Eagle's Medium) treatment on 1% peptide composition. FIG. 5A shows storage modulus data (performed at 1 Pa and 10 rad / s) before and after DMEM treatment of the peptide composition. FIG. 5B shows the increase ratio of the storage elastic modulus after DMEM treatment. FIG. 6A is a photograph of a mixture of RADA16 and DMEM (1: 1 volume ratio). The mixture was low in viscosity and turbid. FIG. 6B shows the mixture after centrifugation. RADA16 (ie, the translucent deposit at the bottom of the centrifuge tube) settled from the mixture. FIG. 7 shows the nanostructures of RADA16, KLD12, and IEIK13 compositions at pH 2-3 and at physiological pH (DMEM). DMEM treatment can form rigid compositions, but mixing with DMEM can precipitate peptides. FIG. 8 shows an exemplary stress sweep test (10 rad / s) of a 1% KLD12 composition at pH = 2.0 and 3.4. FIG. 9 shows an exemplary stress sweep test (10 rad / s) of a 1% IEIK13 composition at pH = 2.1 and 3.7. FIG. 10 shows an exemplary stress sweep test (10 rad / s) of a 1% RADA16 composition at pH = 2.5 and 3.4. The storage elastic modulus of the peptide composition increased with increasing pH. 11A and 11B show exemplary frequency sweep tests of RADA16 at 1 Pa. FIG. 11A is a measurement of 1% RADA16 at pH 2.5 and 3.4. FIG. 11B is a measurement of 2.5% RADA16 at pH 2.5 and 3.4. FIG. 12 is a photograph of 2.5% RADA16 at pH 3.2, 3.4, 3.6 and 4.0. The composition was transparent when the pH level was about 3.5 and was slightly turbid at pH = 3.6. The composition precipitated at pH = 4.0. FIG. 13 shows the nanostructures and / or reorganization of RADA16, KLD12, and IEIK13 at different pH levels with or without shear stress. The predominant interaction can be determined by pH and shear stress. 14-16 show the cell viability (mMSC) of RADA16, IEIK and KLD at selected concentrations, respectively. ^* Means that the cell viability is significantly lower than the cell viability in the column to the left (p-value <0.05). # Means that the cell viability is significantly higher than the cell viability in the column to the left (p-value <0.05). 14-16 show the cell viability (mMSC) of RADA16, IEIK and KLD at selected concentrations, respectively. ^* Means that the cell viability is significantly lower than the cell viability in the column to the left (p-value <0.05). # Means that the cell viability is significantly higher than the cell viability in the column to the left (p-value <0.05). 14-16 show the cell viability (mMSC) of RADA16, IEIK and KLD at selected concentrations, respectively. ^* Means that the cell viability is significantly lower than the cell viability in the column to the left (p-value <0.05). # Means that the cell viability is significantly higher than the cell viability in the column to the left (p-value <0.05). FIG. 17A shows the structure of IEIK13. FIG. 17B shows SEM images of IEIK13 before and after DMEM processing. The IEIK13 fiber after DMEM treatment can be thicker than the fiber before DMEM treatment. FIG. 18 is a bar graph of storage modulus showing the effect of pH on the rheological properties of 2.5% RADA16. The storage elastic modulus was measured at 1 rad / s. FIG. 19 is a bar graph of storage modulus showing the effect of pH on the rheological properties of 1.5% IEIK13. The storage elastic modulus was measured at 1 rad / s. FIG. 20A shows an exemplary flow viscosity test of 1% IEIK13 at pH = 2.1, 3.0, 3.3 and 3.5. FIG. 20B is a bar graph of an exemplary viscosity measurement at a shear rate of 0.003 l / sec. 21A, 21B, 21C and 21D show the time after applying high shear stress to 1% IEIK13 at pH = 2.1, 3.0, 3.3, and 3.5, respectively. The storage modulus measurement as a function of is shown. The horizontal line shows the original storage modulus of 1% IEIK13. 22A and 22B are bar graphs of storage modulus as a function of RADA16 concentration at pH 2.2 and 3.4. FIG. 22A is a measurement before DMEM processing. FIG. 22B is a measurement after DMEM treatment. 23A and 23B are bar graphs of storage modulus as a function of IEIK concentration at pH 2.3 and 3.4. FIG. 23A is a measurement before DMEM processing. FIG. 23B is a measurement after DMEM treatment. FIG. 24 shows a storage modulus measurement as a function of time. FIG. 24 contains 2.5% RADA16 data at pH 2.2, 2.6, 2.8, 3.1 and 3.4. Time sweep tests were performed at 1 rad / sec and 1 Pa (20 mm plate and 500 μm gap distance). During the time sweep test, DMEM was added to the chamber around the measuring plate at time = 0 and the peptide was immersed. FIG. 25 shows a storage modulus measurement as a function of time. FIG. 25 contains 1.5% IEIK13 data at pH 2.3, 2.6, 2.9 and 3.2. Time sweep tests were performed at 1 rad / sec and 1 Pa (20 mm plate and 500 μm gap distance). During the time sweep test, DMEM was added to the chamber around the measuring plate at time = 0 and the peptide was immersed. FIG. 26 shows the nanostructure and / or reorganization of the peptide under low or high salt conditions (ie above critical ionic strength). The method of application (treatment or mixing) of the salt solution can change the nanostructure. Treatment of the peptide with a salt solution can form a rigid gel, but mixing the salt solution with the peptide can cause phase separation. FIG. 27 shows an exemplary frequency sweep test from 1 rad / s to 10 rad / s at 1 Pa. FIG. 27 is a measurement of 1% KLD12 with or without NaCl solution (0.2M ionic strength). FIG. 28 shows an exemplary frequency sweep test from 1 rad / s to 10 rad / s at 1 Pa. FIG. 28 is a measurement of 1% IEIK13 with or without NaCl solution (0.02M ionic strength). FIG. 29 shows an exemplary frequency sweep test from 1 rad / s to 10 rad / s at 1 Pa. FIG. 29 is a measurement of 1% RADA16 with or without NaCl solution (0.7M ionic strength). FIG. 30 is a bar graph of storage elastic modulus at 1 rad / s. 1.0% RADA16 was exposed to NaCl with different ionic strengths from 0 to 1.0 M. FIG. 31 is a bar graph of the storage elastic modulus at 1 rad / s. 1.0% IEIK13 was exposed to NaCl with different ionic strengths from 0 to 0.04 M. FIG. 32A shows a flow viscosity test of 1% IEIK13 at NaCl ionic strengths of 0, 0.01 and 0.02M. FIG. 32B is a bar graph of viscosity at a shear rate of 0.003 l / sec. 33A, 33B and 33C show storage elastic as a function of time after applying high shear stress to 1% IEIK13 at NaCl ionic strengths of 0, 0.01 and 0.02, respectively. The rate measurement is shown. The horizontal line shows the original storage modulus of each 1% IEIK13 composition. FIG. 34 shows an exemplary storage modulus of 1% RADA16 at 1 Pa as a function of NaCl ionic strength before or after DMEM treatment. FIG. 35 shows an exemplary storage modulus of 1% IEIK13 at 1 Pa as a function of NaCl ionic strength before or after DMEM treatment. FIG. 36 shows an exemplary storage modulus of 1% RADA16 with selected salts (NaCl, KCl, MgCl ₂ , and CaCl ₂ ). ^{* Indicates} that G'is significantly higher than the control (without salt) (P <0.05). # Represents that G'is significantly lower than 1% RADA16 with a NaCl ionic strength of 0.15 M (P <0.05). FIG. 37 shows an exemplary storage modulus of 1% RADA16 containing selected salts (NaCl, KCl, MgCl ₂ , and CaCl ₂ ) after DMEM treatment. ^{* Indicates} that G'is significantly higher than the control (without salt, after DMEM treatment) (P <0.05). FIG. 38 shows exemplary storage modulus measurements as a function of time for 1.5% IEIK13, 1.5% KLD12, and 2.5% RADA16 after saline buffer treatment. Time sweep tests were performed at 1 rad / sec and 1 Pa (20 mm plate and 500 μm gap distance). During the time sweep test, at time = 0, saline buffer was added to the chamber around the measuring plate and the peptide was immersed. FIG. 39 shows the nanostructures and / or reorganization of RADA16, KLD12, and IEIK13 at specific ionic strengths. High shear stresses can alter nanostructures and / or reorganization. FIG. 40 shows the storage modulus of 2.5% RADA16 at 1 rad / sec. The addition of NaCl and the increase in pH increased the storage modulus of 2.5% RADA16. 41A, 41B, 41C and 41D show the steps used to prepare peptide compositions with different salts and / or salt concentrations according to Examples 4 and 7. Those of skill in the art will appreciate that similar strategies can be used to analyze peptide compositions with different pH, peptide concentrations, etc., for example. In FIG. 41A, peptide powders were placed using glass vials. In FIG. 41B, the peptide powder was first dissolved in deionized water in a selective fraction of the final volume; optionally, vortex and / or sonication was used to achieve or ensure complete solubilization. .. In FIG. 41C, a concentrated salt solution was added to the top depending on the amount and concentration of the deionized water used. In FIG. 41D, the solutions were mixed, for example by vortexing. 42A, 42B, 42C, 42D, 42E, 42F and 42G are 0.5% RADA16 and 0, 0.005, 0.05, 0.125, 0.250, 0, respectively. .500 and 1M CaCl ₂ photographs of the mixture upright and inverted. FIG. 42E shows an optimal and fully functional gel. FIG. 42F shows a semi-functional gel. FIG. 42G shows a non-functional gel. FIG. 43 shows a storage modulus measurement of 0.5% RADA16 mixed with NaCl, KCl, and CaCl ₂ at concentrations of 0.125, 0.250, and 0.500M. FIG. 44 shows a storage modulus measurement of 2.5% RADA16 containing 2.5% RADA16 and 0.125M CaCl ₂ . 45A and 45B show storage modulus measurements of RADA16 containing 0.125, 0.250, and 0.500M after mechanical perturbation. ^{* Indicates} that the control sample and the perturbation sample are significantly different. FIG. 46 is a storage modulus measurement of 2.5% RADA16 treated with CaCl ₂ and CaSO ₄ as a function of time. FIG. 47A is exemplary rheological data of IEIK13 containing IEIK13 and indigo carmine. FIG. 47B shows the stiffness of IEIK13 containing IEIK13 and indigo carmine. FIG. 47C shows the stiffness of RADA16 and RADA16 containing Ringer's solution. FIG. 47D is an inverted photograph of RADA 16 containing Ringer's solution. FIG. 46E is an inverted photograph of IEIK13 containing IEIK13 and indigo carmine. FIG. 47F is a photograph of IEIK13 placed in a syringe containing indigo carmine. FIG. 48 is a graph of 2.5% RADA16 containing 2.5% RADA16 and NaCl, showing an increase in lung rupture pressure.

Definitions As used herein, the term "drug" can refer to a compound or substance of any chemical class, including, for example, polypeptides, nucleic acids, sugars, lipids, small molecules, metals, or combinations thereof. In some embodiments, the agent is or comprises a natural product in that it is found and / or obtained from nature. In some embodiments, the agent is one or more substances that are artificial in that they are designed, modified, and / or produced, and / or not found naturally, through the movement of the human hand. Or includes. In some embodiments, the agent can be used in isolated or pure form; in some embodiments, the agent can be used in crude form. In some embodiments, the potential agents are provided as a collection or library and, for example, can be screened to identify or characterize the active agent within them. Some specific embodiments of agents that may be used according to the invention include small molecules, antibodies, antibody fragments, aptamers, nucleic acids (eg, siRNA, ThenRNA, DNA / RNA hybrids, antisense oligonucleotides, ribozymes), peptides. Includes peptide mimetics and the like. In some embodiments, the agent is or comprises a polymer. In some embodiments, the agent is not a polymer and / or virtually no polymer is present. In some embodiments, the agent comprises at least one polymer moiety. In some embodiments, the agent is absent or substantially free of any polymer moiety.

As used herein, the term "amino acid" refers in its broadest sense to any compound and / or substance that can be incorporated into a polypeptide chain, eg, through the formation of one or more peptide bonds. In some embodiments, the amino acid has the general structure _H2NC (H) (R) -COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a synthetic amino acid; in some embodiments, the amino acid is a D-amino acid; in some embodiments, the amino acid is an L-amino acid. "Standard amino acid" refers to any 20 standard L-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than the standard amino acid, whether synthetically prepared or obtained from natural origin. In some embodiments, the amino acids in the polypeptide, including carboxy- and / or amino-terminal amino acids, may contain structural modifications as compared to the general structure described above. For example, in some embodiments, amino acids may be modified by methylation, amidation, acetylation, and / or substitution as compared to the general structure. In some embodiments, such modifications may alter, for example, the circulating half-life of a polypeptide containing a modified amino acid as compared to those containing otherwise the same unmodified amino acid. In some embodiments, such modifications do not significantly alter the relevant activity of the polypeptide containing the modified amino acid as compared to those containing otherwise the same unmodified amino acid. As is clear from the context, in some embodiments the term "amino acid" is used to refer to a free amino acid; in some embodiments it is to refer to an amino acid residue of a polypeptide. Used.

As used herein, the term "approximately" or "about" refers to a value that applies to one or more values of interest and is similar to the stated reference value. In certain embodiments, the term "approximately" or "about" is used in either direction (greater than or greater than) of the reference values described, unless otherwise stated or otherwise apparent from the context. 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, Refers to a range of values that fall within the range of 6%, 5%, 4%, 3%, 2%, 1%, or less (unless such numbers exceed 100% of possible values). ..

Two events or substances are "related" to each other when the term is used herein and the presence, level and / or form of one correlates with that of the other. For example, a particular substance (eg, polypeptide, gene signature, metabolite, etc.) may be present, level and / or morphologically (eg, across related populations) with an incidence of disease, disorder, or condition and / or. When correlated with susceptibility, it may be associated with a particular disease, disorder, or condition. In some embodiments, two or more substances are physically "relationship" with each other when they interact directly or indirectly so that they are physically close to each other and / or remain. be". In some embodiments, two or more substances that are physically related to each other are covalently bound to each other; in some embodiments, two or more substances that are physically related to each other are covalently bonded to each other. Not, but non-covalently related, for example, by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetic actions, and combinations thereof.

The term "equivalent" is used herein to describe two (or more) sets of states, situations, individuals or individuals that are sufficiently similar to each other to allow comparison of the results obtained or observed. Used to describe a population. In some embodiments, an equivalent set of conditions, situations, individuals, or populations is characterized by a plurality of substantially identical properties and one or a few different properties. Those skilled in the art will understand that a set of situations, individuals, or populations is equivalent to each other if they are characterized by substantially the same characteristics of sufficient numbers and types, and different sets of situations, individuals, or populations. Differences in the results or observed phenomena under or there justify the reasonable conclusion that they are due to or are signs of diversity in these different characteristics. Those skilled in the art will appreciate that the relative languages used herein (eg, enhanced, activated, reduced, inhibited, etc.) are typically compared made under equivalent conditions. You will understand to point. )

The particular methodology described herein includes a "determining" step. Those skilled in the art who have read this specification will appreciate the use of any of the various techniques available to those of ordinary skill in the art, including the specific techniques expressly referred to herein, for such "decision" steps. You will understand that it can be utilized or achieved through. In some embodiments, the determining step involves manipulating a physical sample. In some embodiments, the determining step comprises, for example, considering and / or manipulating data or information utilizing a computer or other processing device applied to perform the relevant analysis. In some embodiments, the determining step comprises receiving relevant information and / or substance from a source. In some embodiments, the determining step comprises comparing the properties of one or more of the samples or substances with an equivalent reference.

As used herein, the term "gel" refers to a viscoelastic substance whose rheological properties are distinguished from solutions, solids and the like. In some embodiments, the composition is considered to be a gel if its storage modulus (G ′) is greater than its modulus of elasticity (G ″). In some embodiments, the composition is considered to be a gel if there is a chemical or physical cross-linking network (distinguished from entangled molecules in viscous solution) in the solution.

As used herein, the term "in vitro" refers to an event that occurs in an artificial environment, such as in a test tube or reaction vessel, during cell culture, rather than in a multicellular organism.

As used herein, the term "in vivo" refers to an event that occurs within a multicellular organism such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events that occur within a living cell (eg, as opposed to an in vitro system).

As used herein, the term "peptide" is typically relatively short, eg, less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids long. Refers to a polypeptide having an amino acid.

As used herein, the term "polypeptide" refers to any polymer chain of amino acids. In some embodiments, the polypeptide has a naturally occurring amino acid sequence. In some embodiments, the polypeptide has an amino acid sequence that does not occur naturally. In some embodiments, the polypeptide has an amino acid sequence modified in that it was designed and / or produced through the movement of the human hand. In some embodiments, the polypeptide may, or may consist of, a natural amino acid, an unnatural amino acid, or both. In some embodiments, the polypeptide may contain or consist of only natural amino acids, or only non-natural amino acids. In some embodiments, the polypeptide may include D-amino acids, L-amino acids, or both. In some embodiments, the polypeptide may contain only D-amino acids. In some embodiments, the polypeptide may contain only L-amino acids. In some embodiments, the polypeptide is modified or added to one or more amino acid side chains, eg, at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or any combination thereof. It may include one or more pendant groups or other modifications. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegation and the like, including combinations thereof. In some embodiments, the polypeptide may be cyclic and / or may include a cyclic moiety. In some embodiments, the polypeptide is not cyclic and / or does not contain any cyclic moieties. In some embodiments, the polypeptide is linear. In some embodiments, the polypeptide may or may be a staple polypeptide. In some embodiments, the term "polypeptide" can be added to the name, activity, or structure of a reference polypeptide; in such cases, to refer to a polypeptide that shares a related activity or structure. As used herein, and thus can be considered members of the same class or family of polypeptides. For each such class, the present specification provides exemplary polypeptides within the class in which the amino acid sequence and / or function is known, and / or those skilled in the art are aware; some embodiments. So, such exemplary polypeptides are reference polypeptides for a polypeptide class or family. In some embodiments, members of the polypeptide class or family exhibit significant sequence homology or identity with the reference polypeptide of the class; in some embodiments with all polypeptides in the class). , Share common sequence motifs (eg, characteristic sequence factors), and / or share common activity (in some embodiments, equivalent levels, or within a predetermined range). For example, in some embodiments, the member polypeptide is at least about 30-40% and about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%. , 95%, 96%, 97%, 98%, 99% or higher, indicating the overall degree of sequence homology or identity with the reference polypeptide, and / or 90%. Or at least one region exhibiting very high sequence identity, often higher than 95%, 96%, 97%, 98%, or 99% (eg, characteristic sequences in some embodiments). Containing areas that can or may contain factors). Such conservative regions usually contain at least 3-4, often up to 20 or more amino acids; in some embodiments, the conservative region is at least one sequence. Contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive amino acids. In some embodiments, the useful polypeptide may, or may consist of, a fragment of the parent polypeptide. In some embodiments, a useful polypeptide may contain or consist of multiple fragments, each of which is the poly of interest such that the polypeptide of interest is a derivative of its parent polypeptide. Fragments that are directly bound within the parent are found spatially separated within the polypeptide of interest, with spatial arrangements that differ from those found within the peptide. And / or vice versa, the fragments may be present in the polypeptide of interest in a different order than within the parent).

As used herein, the term "reference" describes a standard or control to which a comparison is made. For example, in some embodiments, the agent, animal, individual, population, sample, sequence or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, the reference or control is tested and / or determined substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium of expression. Typically, as will be appreciated by one of ordinary skill in the art, the reference or control will be determined or characterized under the same conditions or circumstances as those under evaluation. Those of skill in the art will appreciate that, in the presence of sufficient similarity, the dependence and / or comparison on a particular possible reference or control is justified.

The term "self-assembling" is used herein to specify that, for example, a solution containing them (eg, an aqueous solution) can spontaneously self-associate with a structure under appropriate conditions so as to develop gel properties. Used for the polypeptide of. In some embodiments, the interactions between and within the individual self-assembled polypeptides within the composition are reversible so that the composition can reversibly transition between the gel and solution states. be. In some embodiments, self-assembling (and / or de-assembling) is one or more environmental triggers (eg, pH, temperature, ionic strength, molar osmolality, osmolality, applied pressure). Responsive to one or more changes, such as applied shear stress. In some embodiments, the composition of the self-assembled polypeptide is characterized by a detectable β-sheet structure when the polypeptide is in an organized state.

[Detailed description of a particular embodiment]
According to one or more embodiments, the invention provides a formulation of a particular peptide that can provide enhanced utility and improved performance as compared to other formulations of the same peptide. In some embodiments, the disclosed formulations may provide different or unique properties that may address previously unmet requirements, eg, associated with various studies and / or clinical applications. In some embodiments, certain desirable properties of the peptide formulation provided are by increasing the pH level of the formulation compared to the standard or reference formulation of the peptide, and / or in the standard or reference formulation. It is provided by adding one or more salts to the pharmaceutical product in comparison with the type and / or amount of the salt of. In some embodiments, the provided formulation is characterized by more stable hydrogel formation and / or other attributes compared to the standard or reference formulations described herein.

Peptides According to one or more embodiments, the peptide composition may comprise an amphipathic polypeptide having about 6 to about 200 amino acid residues. In certain embodiments, it may have a length of at least about 7 amino acids. In certain embodiments, the polypeptide can have a length of about 7 to about 17 amino acids. In certain embodiments, the polypeptide can have a length of at least 8 amino acids, at least about 12 amino acids, or at least about 16 amino acids.

In some embodiments, as will be understood in the art, amphipathic polypeptides are those in which the sequence comprises both hydrophilic and hydrophobic amino acids. In some embodiments, such hydrophilic and hydrophobic amino acids may be attached alternately such that the peptide has an alternating hydrophilic and hydrophobic amino acid sequence. In some embodiments, the polypeptide for use according to the present disclosure has an amino acid sequence comprising or consisting of repeating units of the sequence Arg-Ala-Asp-Ala (RADA). In some embodiments, the polypeptide for use according to the present disclosure has an amino acid sequence comprising or consisting of repeating units of the sequence Lys-Leu-Asp-Leu (KLDL). In some embodiments, the polypeptide for use according to the present disclosure has an amino acid sequence comprising or consisting of repeating units of the sequence Ile-Glu-Ile-Lys (IEIK).

In some embodiments, the peptides for use according to the present disclosure may be generally self-assembled and / or exhibit a β-sheet structure in aqueous solution under certain conditions.

In some embodiments, the peptides for use according to the present disclosure are amino acid sequences: Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-. It has Ala (ie, RADA16, aka [RADA] 4; SEQ ID NO: 1). In some embodiments, the peptides for use according to the present disclosure are amino acid sequences: Lys-Leu-Asp-Leu-Lys-Leu-Asp-Leu-Lys-Leu-Asp-Leu (ie, KLDL12, aka [i.e. KLDL]) 3 aka KLD12; SEQ ID NO: 2). Peptides for use according to the present disclosure are amino acid sequences: Ile-Glu-Ile-Lys-Ile-Glu-Ile-Lys-Ile-Glu-Ile-Lys-Ile (ie, IEIK13, aka (IEIK) 3I; sequence. It has the number 3).

Those skilled in the art who read this specification will appreciate that any variety of other peptides may be used in place of the practice of the present invention. In some embodiments, for example, published US patent application US2009 / 0111734 A1, published US patent application US2008 / 0032934 A1, published US patent application US2014 / 0038909 A1, issued US patent US7,846891. B2, U.S. Patent US7,713923 B2, U.S. Pat. No. 6,670483 B2, one or more peptides described herein, the related content thereof, is incorporated herein by reference.

In some embodiments, the peptide for use according to the invention has an amino acid sequence comprising or consisting of a sequence represented by one of the following formulas:
((XY) _l- (ZY) _m ) _n ... Equation (a)
((YX) _l- (YZ) _m ) _n ... Equation (b)
((ZY) _l- (XY) _m ) _n ... Equation (c)
((YZ) _l- (YX) _m ) _n ... Equation (d)
Here, X represents an acidic amino acid, Y represents a hydrophobic amino acid, and Z represents a basic amino acid, where l, m and n are all integers (n (l + m) <200) (1 ≦). n ≦ 100)).

Composition In some embodiments, the peptide composition according to the present disclosure may be characterized by specific rheological and / or optical properties. In some embodiments, such rheological properties may include one or more such as gelling kinetics, reversible organizing properties, storage modulus, viscosity and the like. In some embodiments, one or more rheological properties can be tested and / or determined (eg, measured); in some embodiments, one or more rheological properties are evaluated by visual observation. Can be done.

In some embodiments, the relevant optical properties may include one or more, such as the degree of transparency, optical transparency, and the like. In some embodiments, one or more optical properties can be tested and / or determined (eg, measured); in some embodiments, one or more optical properties are visually observed. Can be evaluated by. In some embodiments, the optical transparency of a particular composition can be described as clear, slightly opaque, or opaque. In some embodiments, the provided composition is transparent.

In some embodiments, the provided composition is characterized by a particular level of stiffness. In some embodiments, stiffness is assessed by determining the storage modulus. As will be appreciated by those skilled in the art, storage modulus and stiffness generally have a positive correlation; that is, a higher modulus of storage correlates with a higher stiffness.

In some embodiments, the provided composition is characterized by a particular gelling property (eg, a particular degree of gelation within a particular period of time). In some embodiments, the provided composition is characterized by a substantially complete gelation within a period ranging from about 10 seconds to about 48 hours.

In some embodiments, the provided composition is characterized by a particular degree of gelation and / or restoration of other substances and / or rheological properties. For example, in some embodiments, when the gelled provided compositions are subjected to disintegration, they are within a specific time period (eg, within the range of about 10 seconds to about 48 hours). Shows the ability to regel into a gel whose mechanical and / or rheological properties are reasonably equivalent to those of the original gel.

In some embodiments, the provided composition is characterized by its ability to support cell proliferation and / or viability.

In some embodiments, the properties of one or more substances (eg, leology) of the peptide composition described and / or used herein are, for example, peptide identity (eg, amino acid sequence, hydrophobic). Degree), peptide concentration, pH, ionic strength (eg, salt concentration), ion identity, etc., and combinations thereof.

Peptide Concentration According to one or more embodiments, the rheological properties of the peptide compositions described herein can be adjusted by selecting the peptide concentration. The present disclosure allows, for example, the selection and / or production of peptide compositions with certain desired properties, which may be particularly preferred for a particular application or use of the composition, through selection and / or regulation of peptide concentration. Define the parameters.

For example, the present disclosure shows that, especially for many peptides, compositions, stiffness increases substantially linearly with peptide concentration. Moreover, as described herein, certain peptide compositions exhibited shear thinning properties above critical stress levels. Furthermore, the present disclosure shows that the rheological properties achieved at a particular peptide concentration can vary depending on the identity of the peptide. For example, the storage modulus of 1.5% KLD12 was found to be similar to that of 2.5% RADA16. It was found that the storage modulus of 1% IEIK13 was similar to that of 2.5% KLD12 and higher than that of 2.5% RADA16. In general, the order of rheological intensities in the compositions tested in this example is IEIK13> KLD12> RADA16, and the IEIK13 compositions show greater rheological intensities than the KLD12 compositions, as well. It showed greater rheological intensity than the composition of RADA16 (in each case, when the peptide concentrations were the same).

In some embodiments, the peptide concentration in the peptide composition for use according to the invention is at least 0.05%, at least 0.25%, at least 0.5%, at least 0.75%, at least 1. 0% or more. In some embodiments, the peptide concentration in the peptide composition for use according to the invention is less than 5%, less than 4.5%, less than 4%, less than 3.5%, less than 3%, or more. There are few. In some embodiments, the peptide concentration in the peptide composition for use according to the invention is in the range between about 0.5% and about 3%. In some embodiments, the peptide concentration in the peptide composition for use according to the invention is in the range between about 0.5% and about 2.5%. In some embodiments, the peptide concentration in the peptide composition for use according to the invention is in the range between about 1% and about 3%. In some embodiments, the peptide concentration in the peptide composition for use according to the invention is in the range between about 1% and about 2.5%. In some embodiments, the peptide concentration in the peptide composition for use according to the invention is about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about. 3% or higher. In some specific embodiments where the peptide is RADA16, the peptide concentration in the peptide composition of the invention is in the range of about 0.05% to about 10%.

In some specific embodiments where the peptide is KLD12, the peptide concentration in the peptide composition of the invention is in the range of about 0.05% to about 10%.

In some specific embodiments where the peptide is IEIK13, the peptide concentration in the peptide composition of the invention is in the range of about 0.05% to about 10%.

pH
The present disclosure shows, among other things, that pH can affect the properties of peptide compositions. As described herein, optimizing the pH of the peptide composition can improve the mechanical strength so that the peptide composition can be used in a variety of clinical applications. Example 3 in the present disclosure shows the details of a specific specific embodiment.

According to one or more embodiments, the peptide composition provided is higher (eg, significantly) than the pI of the relevant peptide and / or the pI of what would be obtained if the peptide were solubilized in water. Can have a high) pH. In some embodiments, the properties of the peptide composition can be adjusted by pH. For example, in some embodiments, at a pH in the range of about 2.5 to about 4.0, the stiffness and / or viscosity of the peptide composition will be appropriate reference composition (eg, the same peptide at the same concentration in water). ) Can be increased compared to that of.

In some embodiments, the peptide composition may comprise a peptide and a solvent, typically an aqueous solvent, and the pH may be adjusted via a pH regulator such as a base or acid. In some embodiments, the peptide composition comprises a peptide and buffer.

In some embodiments, the pH adjusted peptide composition comprises one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, sodium acetate, sodium sulfide, DMEM and / or PBS. obtain.

In some embodiments, an automatic titration device may be performed for pH adjustment.

In some embodiments, the provided composition has and / or maintains a pH higher (eg, substantially higher) than the pI of the associated peptide. In some embodiments, the provided composition has and / or maintains a higher (eg, substantially higher) pH than that of an aqueous solution of the same peptide at the same concentration. In some embodiments, the provided composition has and / or maintains a pH lower than the pH at which the composition is turbid or turbid.

In some embodiments, the provided composition is characterized by a pH of about 2.5-4.0 or higher; in some embodiments, the provided composition is physiologic. Characterized by a pH closer to the target pH. In some embodiments, the provided composition has a pH in the range of about 3.0-4.0. In some embodiments, the composition provided is about 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, about 3.1, about 3.2, about. It has a pH of 3.3, about 3.4, about 3.5, or higher, or higher. In some embodiments, the provided compositions are about 4.3, about 4.2, about 4.1, about 4.0, about 39. , About 3.7, about 3.6, about 3.5, about 3.4, or below, or below.

In some embodiments, the elevated pH compositions described herein (ie, compositions having a pH of about 2.5 or higher) are suitable reference compositions (eg, at the same concentration). , Equivalent compositions of the same peptide, optionally having the same salts but different pH), characterized by greater rheological rigidity and / or improved gelling properties. In some embodiments, the elevated pH composition is useful in a wider range of applications than the corresponding reference composition at a lower pH.

The present disclosure can significantly increase the stiffness of the IEIK13 composition, while not increasing the stiffness of the RADA16 and KLD12 compositions, especially at elevated pH 3.5 or lower, in some embodiments. Show that. Without wishing to be bound by any particular theory, the present disclosure shows that the different behaviors of peptide compositions at pH 3.5 or lower are pKa (pKa = 3) of aspartic acid (D) in RADA16 and KLD12. It is proposed that it may be related to .71) and glutamic acid (E) (pKa = 4.15) of IEIK13.

When the pH is higher than the pKa of aspartic acid (D) and glutamic acid (E), the acidic groups in the peptide chain are almost negatively charged. Negatively charged groups induce intramolecular or intramolecular, attractive intermolecular interactions with positively charged groups within the peptide chain (ie, arginine (R) of RADA16 and lysine (K) of IEIK13 and KLD12). And rather than forming nanofibers (ie, transparent viscous compositions), they form larger aggregates that are translucent or opaque (ie, above their cloud point), giving them the possibility of phase separation.

If the pH is lower than the pKa of aspartic acid (D) and glutamic acid (E) but close to that pKa, the more aggregated negatively charged groups may induce a stronger attracting charge-to-charge interaction with the positive group. .. The composition may maintain the shape of the nanofibers so that the rigidity is increased.

A particular exemplary peptide composition having a pH of about 3.5 is shown in Table 2. Such compositions, which are considered "compositions with elevated pH" herein, have improved performance in various applications (eg, the relevant reference compositions described herein, including examples. It may provide a composition (compared to a composition with a lower pH, such as one, otherwise equivalent). The mechanical strength and pluripotency of the peptide composition can be enhanced by elevated pH.

According to one or more embodiments, pH can affect gelation kinetics (eg, response time to initiate gelation). The effect of pH on gelled kinetics can be evaluated to identify the optimum pH for the peptide compositions described herein.

In some embodiments, the peptide composition can gel faster at higher pH. For example, as described herein, the unadjusted IEIK13 composition shows an immediate increase in storage modulus, while the unadjusted (pH 2.2) RADA16 composition is the first 13. Does not show an increase in storage modulus for a second. Upon pH adjustment, both IEIK13 and RADA16 show an immediate increase in storage modulus due to rapid gelation, as shown in FIGS. 24 and 25.

Ionic Strength The present disclosure shows that ionic strength can change the rheological properties of peptide compositions. Increasing the ionic strength of a peptide composition can generally improve mechanical properties for various clinical applications. For example, as described herein, the effect of ionic strength on the properties of the peptide composition may be evaluated to identify the optimal ionic strength for the peptide composition described herein.

In some embodiments, the stiffness, viscosity, and / or gelling kinetics of the peptide composition can be increased at ionic strengths in the range of about 0.0001M to about 1.5M. In some embodiments, the peptide composition can be adjusted by ionic strength.

According to one or more embodiments, the ionic strength of the peptide composition can be regulated by one or more of the common salts, including but not limited to NaCl, KCl, MgCl ₂ , CaCl ₂ and CaSO ₄ . .. Common salts consist of cations and anions. In some embodiments, the cation may be selected from the group comprising ammonium, calcium, iron, magnesium, potassium, pyridinium, quaternary ammonium, and sodium. In some embodiments, the anion may be selected from the group comprising acetate, carbonate, chloride, citrate, cyanide, flolide, nitrate, and phosphate.

According to one or more embodiments, the addition of one or more salts or salt solutions can be carefully controlled when the ionic strength approaches optimal levels (eg, maximum stiffness). In some embodiments, pure water may be added if the ionic strength is higher than desired. The addition of one or more salts or salt solutions can be controlled to adjust the osmotic pressure to hypotonic, isotonic or hypertonic depending on its application.

In some embodiments, as an example, certain salt buffers such as NaCl, KCl, MgCl ₂ , CaCl ₂ and DPBS (Dalvecolinic acid buffered saline) are used to regulate the ionic strength of a particular peptide composition. Water, 10x) can be added.

In some embodiments, the provided composition comprises one or more salts, the identity and / or concentration of which is critical ionic strength (below which material precipitation of the peptide is seen). To maintain. In some embodiments, material precipitation is believed to occur when the liquid composition is turbid (eg, evaluated by visual inspection). Thus, in some embodiments, the provided composition is not turbid and has ionic strength that is otherwise opaque and otherwise comparable (eg, the same peptide of the same concentration). ..

In some embodiments, the provided composition is compared to a suitable reference composition (eg, a composition of the same peptide at the same concentration and pH, but with different salts or the same salt at different concentrations). And is characterized by increased ionic strength. In some embodiments, the provided composition is characterized by ionic strength close to or at physiological strength. In some embodiments, the compositions described herein are characterized by greater rheological stiffness and / or improved gelling properties compared to suitable reference compositions with different ionic strengths. In some embodiments, the provided compositions are suitable for use in a wider range of applications than the corresponding reference compositions with different (eg, lower) ionic strengths.

According to one particular embodiment, a peptide composition comprising IEIK13, KLD12, or RADA16 in a salt solution is provided, the composition of which is an ion different from the reference composition of the relevant peptide dissolved in water. It has strength and exhibits one or more improved material (eg, leology) properties compared to its reference composition. In some embodiments, the provided composition is more rigid than the associated reference composition. In some embodiments, the provided compositions have increased ionic strength as compared to the reference composition, but still have ionic strength lower than their salt critical point.

Without wishing to be bound by any particular theory, the present disclosure proposes that the properties of peptide compositions with increased ionic strength may be related to the solubility of the peptide. The solubility of the self-assembled peptide at pH levels of about 2-4 is almost high enough to make a clear and homogeneous peptide composition. Increasing the ionic strength around the peptide chain reduces the solubility of the peptide. If the solubility of the peptide is low so that the composition becomes turbid, this condition may be referred to as the critical point. If the increased ionic strength is below but close to its critical point, the peptide can induce stronger hydrophobic interactions and increase stiffness. If the peptide solubility drops below its critical point (ie, high ionic strength), the peptide composition can be translucent or opaque (ie, above its cloud point) and can precipitate (ie, phase). Separation). Peptides cannot form nanofibers that produce clear, viscous solutions. Random hydrophobic interactions can predominate over hydrophobic interactions that produce self-assembled nanofibers at high ionic strength due to the salting out effect. Random intramolecular and / or intermolecular aggregates can cause phase separation.

According to one or more embodiments, the critical ionic strength can vary depending on the salt and peptide identity. The relationship between solubility and salinity can be expressed by Cohen's equation:
log S = B-KI
When S is the solubility of the peptide, B is the peptide-specific constant, K is the salt-specific constant, and I is the ionic strength of the salt. B correlates with pH and temperature. K correlates with pH.

In some embodiments, the solubility of the peptide may depend on the salting out constant K and the ionic strength I if the temperature and pH are constant (ie B is constant). Higher K and I result in lower peptide solubility. At constant pH and temperature, K is determined by ionic identity in the salts. Generally, the order of the constant K among the four salts is NaCl>KCl> MgCl ₂ = CaCl ₂ .

According to one or more embodiments, the solubility of a peptide can be determined by the amino acid sequence (eg, the composition of hydrophilic and hydrophobic amino acid residues in the peptide). Peptides with a relatively high hydrophobic amino acid content (eg, IEIK13) are typically sparingly soluble in aqueous solvents. Such peptides are often characterized by strong hydrophobic interactions between self-assembled peptide chains, resulting in high stiffness. As shown herein, compositions of such peptides may exhibit a dramatic increase in stiffness with the addition of small amounts of salt. In contrast, peptides with relatively low hydrophobic amino acid content (eg, RADA16) have high solubility in aqueous solvents. These peptides typically have weak hydrophobic interactions between self-assembled peptides, resulting in low stiffness. The stiffness of the composition of such peptides does not increase significantly even with the addition of large amounts of salt. Consistent with this model, the present disclosure shows the order of critical ionic strength (eg, when the composition becomes turbid) in three specific exemplified peptides parallel to relative hydrophobicity: RADA16 (0). .9 to 1.2M)> KLD12 (0.3 to 0.4M)> IEIK13 (0.03 to 0.04M).

According to one or more embodiments, the ionic strength of the peptide composition can affect its gelling kinetics. In some embodiments, the increased ionic strength may promote gelation of the peptide composition. The ionic strength required for gelation may depend on salt and / or peptide identity. For example, when RADA16, KLD12, and IEIK13 peptides were exposed to saline buffer (ie 0.15M NaCl, equivalent to isotonic fluid), only gelation of IEIK13 was initiated. RADA16 and KLD12 showed no gelation or were negligible. These findings may reflect the decrease in peptide solubility with increased ionic strength. IEIK13 is more sensitive to ionic strength than RADA16 and KLD12 described above.

In some embodiments, the ionic strength of the peptide composition is, for example, the first aggregate formed (eg, nanofibers) due to the (typically hydrophobic) peptide-to-peptide interaction, eg, mixing and / or stirring treatment. ) Can affect its resilience properties after it has collapsed.

Effects of Combined pH and Salt The present disclosure shows that simultaneous adjustment of pH and ionic strength (eg, by exposure to physiological conditions) can alter the rheological properties of the peptide composition. For example, as described herein, the increased pH level and ionic strength resulting from the inclusion of cell culture medium in the provided peptide composition is a variety of properties of such composition (eg, rheological properties). ) Can be affected.

In some embodiments, the stiffness, viscosity and / or gelling kinetics of the peptide composition can be increased under physiological conditions. In some embodiments, the properties of the peptide composition can be adjusted by a combination of pH and ionic strength.

Without wishing to be bound by any particular theory, the present disclosure states that there are two major intermolecular interactions associated with stiffness: hydrophobic interactions and charge-to-charge interactions of peptide compositions. suggest.

First, hydrophobic and repulsive electrostatic interactions are the main driving force for forming viscous solutions through the formation of β-sheet nanofibers at low pH. These interactions are expected to be significant at low pH when most of aspartic acid and glutamic acid are protonated without negative charge and most of arginine and lysine are positively charged. Peptide molecules self-assemble to form nanofibers due to hydrophobic interactions, while the surface of the nanofibers is hydrated due to repulsive electrostatic interactions between the peptide molecules.

In some embodiments, the stiffness of the peptide composition around a pH level of about 2-3 should largely correlate with their hydrophobicity. IEIK13 has 7 isoleucine groups (strong hydrophobic groups), KLD12 has 6 leucine groups (strong hydrophobic groups), and RADA16 has 8 alanine groups (weak hydrophobic groups). Has. IEIK13 has a higher storage modulus than KLD12 and RADA16 at the same pH and concentration.

As shown in FIG. 17B, when the IEIK13 molecules in aqueous solution are treated with simulated body fluid (eg, DMEM), their fibrous structure becomes thicker. Thicker fiber structures can result from increased hydrophobic interactions at physiological pH and osmotic pressure between adjacent nanofibers.

Hydrophobic interactions can induce nanofiber formation in an aqueous environment, producing viscous compositions. After application of high shear stress (ie, reduced viscosity and stiffness), the peptide can also reshape nanofibers to restore their properties. Therefore, peptides exhibit thixotropic properties at pH 2-3. Peptide compositions slowly restore their original properties when the applied shear stress is removed.

Second, attracting charge-to-charge interactions can occur simultaneously with existing hydrophobic interactions under physiological conditions. If the pH around the peptide molecule changes from acidic to neutral, the existing hydrophobic interactions cannot be disrupted. Negative and positive charge groups induce further intramolecular interactions between the attracting charges so that the peptide composition may be more rigid, as shown in FIG.

However, when peptide aggregates under physiological conditions are exposed to high shear stress, the peptide aggregates disintegrate into peptide aggregates. As shown in FIG. 7, this is an irreversible process.

For example, when 0.5 mL of DMEM was mixed with 0.5 mL of 2% RADA16 by pipetting several times, RADA16 did not form a clear, viscous peptide aggregate (ie, a turbid, less viscous emulsion). .. When the mixture was centrifuged at 2500 rpm for 5 minutes, the turbid phase separation of RADA16 settled from the mixture. In this case, the peptide aggregate (ie, initially formed through hydrophobic interactions) could be disrupted during the mixing process. The attracting charge-to-charge interactions prevailed over the hydrophobic interactions. It induces the formation of random intramolecular and intermolecular aggregates. The phase separation is shown in FIG.

According to one or more embodiments, IEIK13, KLD12, or RADA16 can be dissolved in salt buffer (eg NaCl) and their pH can be increased by alkaline salt buffer (eg NaOH). Their salt ionic strength can be below their salt critical point. Their pH can be from about 2.5 to about 4.0. The composition may have increased stiffness and viscosity as compared to a suitable reference composition of the same peptide at the same concentration.

In some embodiments, physiological conditions (eg, elevated pH and salt ionic strength) can promote gelation of the peptide composition. The accelerated gelation of IEIK13 under physiological conditions may be associated with two driving forces, namely increased pH and ionic strength. The accelerated gelation of RADA16 under physiological conditions may have only one driving force, i.e. increased pH. In some embodiments, accelerated gelation of the peptide composition in body fluids can generally improve its function and response time to various clinical applications.

Cell compatibility According to one or more embodiments, the peptide compositions provided are generally associated with high cell viability.

In some embodiments, KLD12 and IEIK13 may have similar or higher cell viability compared to RADA16. The overall cell viability sequence in these peptide compositions was KLD12> IEIK13> RADA16. In some embodiments, peptide compositions may have cell viability greater than 80% when their concentration is about 0.75% or less. In some embodiments, cell viability may be reduced if the peptide concentration is higher than 0.75%.

Shape [In some embodiments, the peptide composition according to the invention is in the form of a dry powder, solution, gel (eg, hydrogel), or any combination thereof.

In some embodiments, the dry powder composition comprises an appropriate amount of peptide and is selected in volume of solvent (eg, aqueous solvent, optionally one or more salts and / or one or more pH). A solution of the desired concentration is obtained upon addition of (including a regulator). In some embodiments, the dry powder composition comprises the appropriate type and relative amount of salt and peptide, with selected volumes of solvent (eg, aqueous solvent, optionally one or more additional salts and / /). Or when added (including one or more pH adjusters), a solution of the desired peptide concentration and ionic strength described herein is obtained. In some embodiments, the dry powder composition comprises the appropriate type and relative amount of pH regulator and peptide, with selected volumes of solvent (eg, aqueous solvent, optionally one or more salts and). Upon addition of / or including one or more additional pH regulators) results in a solution of the desired peptide concentration and pH described herein. In some embodiments, the dry powder composition comprises the appropriate type and relative amount of pH adjuster, salt, and peptide and is selected volume of solvent (eg, aqueous solvent, optionally one or more). With the addition of additional salts and / or one or more additional pH adjusters), a solution of the desired peptide concentration, pH, and / or ionic strength described herein is obtained.

In some embodiments, the provided composition is housed in a container (eg, syringe, vial, well, etc.). In some embodiments, the container is a graduated container in that it includes a volume indicator. In some embodiments, the container is suitable for connection to a delivery device such as a cannula or syringe. In some embodiments, the container is sealed in a manner (eg, intrusive covering) that allows the addition and / or removal of a fluid (eg, liquid) substance without removing the seal. ..

Applications In some embodiments, the present disclosure provides a system for selecting peptide compositions for use in a particular application. The effects of peptide identity, peptide concentration, pH, salt identity and / or salt concentration, as described herein, depend on the properties (and thus the usefulness of the particular peptide composition for a particular application). Can influence.

To provide a few examples, peptide compositions with higher stiffness are generally more suitable for applications characterized by hemostasis, tissue plugs, anti-adhesions, or specific tissue regeneration. Peptide compositions with a faster gelation time typically require or prefer a gelation time of less than about 1 minute to about 1 hour for specific tissue plug applications, hemostasis, tissue plugs, anti-adhesions. , Or drug transport, vascular plugs, etc.) may be particularly suitable. Peptide compositions with faster restoration times may be particularly suitable for hemostasis, tissue plugs, or vascular plugs.

As shown herein, self-organizations that are extremely useful in a variety of in vivo and in vitro contexts, including, for example, as cell scaffolds, fluid transfer barriers, hemostatic agents, void fillers, and more. Chemical peptide compositions are provided. Different such compositions described herein may be more useful in different contexts.

For example, the context for administration of a peptide composition (eg, as a hemostatic agent) during a surgical procedure is that the composition remains substantially liquid for a suitable period of time for administration to the surgical site and then rapid. Can benefit from gelling kinetics, which allows to gel to form a stable, preferably transparent, relatively rigid gel (so that surgical procedures can be facilitated). ..

Provided by way of example, as described herein, in some embodiments, the IEIK13 composition is particularly in various biomedical applications where, for example, certain stiffness and rapid gelation are required. Can be useful. The present disclosure shows that a particular IEIK13 composition is characterized by a rapid recovery rate (eg, the fastest self-assembly) and / or relatively high stiffness after application of high shear stress. The present disclosure also shows that certain IEIK13 compositions can exhibit particularly useful (eg, high) stiffness when in contact with physiological media.

The present disclosure also shows that certain KLD12 compositions may be particularly useful, for example, where simple injection is required with high final stiffness. In some embodiments, the self-assembled nanofibers of KLD12 can be reverse-assembled by high shear stress and then slowly reassembled.

The present disclosure also shows that certain KLD12 compositions may be particularly useful when high cell viability is required. In some embodiments, the concentration of KLD12 may be increased to provide the stiffness required for a particular application.

Example 1: Optical Transparency of a Specific Reference Peptide Composition In this example, the indicated peptide is dissolved in water, and the optical transparency and phase stability of the specific reference peptide composition (ie, incomplete phase separation). Existence) is shown. In some embodiments, the optical clarity (and / or phase stability) of the provided peptide composition is assessed relative to that of such a reference composition. In some embodiments, the composition provided with a particular peptide at a particular concentration has at least as good optical transparency and optical transparency as that of a reference composition of the same peptide in the same composition dissolved in water. / Or indicates phase stability.

As can be seen with respect to Table 1, various reference peptide compositions were prepared that exhibited optical transparency (and also phase stability) over various peptide concentrations. In particular, peptide compositions at 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5% or higher concentrations have transparent optical properties and It showed the absence of phase separation.

Example 2: Rheological Properties of Peptide Compositions as a Function of Concentration This Example shows the effect of peptide concentration on the rheological properties of a particular peptide composition. In some embodiments, the rheological properties may have a linear correlation with the peptide concentration.

In some embodiments, the peptide composition having a particular desired stiffness has a particular peptide concentration determined using a mathematical model (ie, a modulus trend-line equation). Can be formulated to have. The pharmaceutical composition charts of the peptide compositions may relate their concentration in deionized water to their unique storage modulus, eg, as shown in Table 3A below. From such charts, one of ordinary skill in the art will formulate a peptide composition with the desired rheological properties by selecting the particular peptide and the appropriate peptide concentration so that the composition to be formulated has the desired stiffness. obtain.

For example, as described herein, the order of rheological intensities of a peptide composition comprising a RADA16, KLD12, or IEIK13 peptide is generally indicated as IEIK13> KLD12> RADA16.

This example further illustrates the measurement of various rheological properties of a particular peptide composition at selected concentrations using a rheometer (AR500, TA Instruments) equipped with a 40 mm plate. The peptide composition (700 μL) was placed on a rheometer plate and the excess composition was gently removed with a Kimwipe. Measurements were taken after a 2 minute relaxation time at 37 ° C. Using a plate placed with a measurement geometric gap of 300 μm, the storage modulus, loss modulus, and viscosity (η') were measured at 37 ° C., and the stress sweep test was performed with a vibration stress of 0.1 Pa to 1000 Pa. The measurement was performed at an angular frequency of 10 rad / s.

The results are shown in FIGS. 2-4 for RADA16, IEIK13, and KLD12. As shown in FIGS. 2 to 4, when the vibration stress was less than about 10 to 200 Pa, the peptide composition showed almost the elastic modulus of the plateau. They all had a dramatic reduction in elastic modulus at certain yield oscillation stresses. Within the tested concentration range of at least about 1% to about 2.5%, the peptide composition showed a linear increase in storage modulus with increasing concentration (R2 on the linear trend line was 0.971. Between 0.992). Certain peptide compositions exhibited shear thinning properties above critical stress levels.

The rheological results determined for the various peptide compositions tested in this example are shown in Table 3. As can be seen, the storage modulus of 1.5% KLD12 was found to be similar to that of 2.5% RADA16. The storage modulus of 1% IEIK13 was found to be similar to that of 2.5% KLD12 and higher than that of 2.5% RADA16. In general, the order of rheological intensities among the compositions tested here was IEIK13> KLD12> RADA16.

Example 3: Rheological Properties of Peptide Compositions as a Function of pH This Example shows the effect of pH on the rheological properties of a particular peptide composition. In some embodiments, pH can be a control parameter that affects the stiffness, viscosity, and / or restoration time of the peptide composition.

Table 2 below shows the pH concentrations found for the reference composition in which the indicated peptides are solubilized in water at the indicated concentrations.

In this example, the pH level of the peptide composition was adjusted, for example, by adding 0.1N NaOH to 2 mL of the 2.5% peptide composition. The pH and appearance of the adjusted composition were observed. If the pH level was higher than desired, an acid salt was added.

The results are shown in Table 5. In particular, increased pH (ie, up to about 3.5 or less) did not change the clear color of the RADA16, IEIK13, and KLD12 compositions, while increasing their apparent stiffness. For certain compositions, when the pH level of the peptide composition is above 3.5 (RADA16 and KLD12) or above 3.7 (IEIK13), the peptide composition begins to phase separate (ie, become turbid). ). In some embodiments, the peptide composition provided is from about 3.0 to about 3.7 (specifically for IEIK3), or from about 3.0 to about 3.5 (specifically RADA16 and). / Or have a pH in the range of (with respect to KLD12).

Rheological properties of certain peptide compositions were observed before and after adjusting their pH levels to 3.4 (RADA16 and KLD12) or 3.7 (IEIK13). Rheological properties of the peptide were evaluated using a rheometer (AR500, TA Instruments) equipped with a 40 mm plate. Specifically, the peptide composition (700 μL) was placed on a rheometer plate and the excess composition was gently removed with a Kimwipe. Measurements were taken after a 2 minute relaxation time at 37 ° C. The results of the stress sweep test are shown in FIGS. 8 to 11. The RADA16, KLD12 and IEIK13 compositions at elevated pH were more rigid than those at 2.5 (RADA16), 2.0 (KLD12), and 2.1 (IEIK13).

The storage modulus of a particular peptide composition at selected pH levels was evaluated using a rheometer (DHR-1, TA Instruments) equipped with a 20 mm plate. The storage modulus of the RADA16 and IEIK13 compositions increased with increasing pH up to 3.4. The storage modulus determined for the peptide composition tested is shown in FIG. 18 for RADA16 and FIG. 19 for IEIK13, respectively.

The viscosity of the 1% IEIK13 composition at the selected pH level was evaluated using a rheometer (DHR-1, TA Instruments) equipped with a 20 mm plate. The viscosity of the IEIK13 composition increased with increasing pH up to 3.5. The IEIK13 composition exhibited typical shear thinning properties. The results are shown in FIGS. 20A and 20B.

After applying high shear stress to the 1% IEIK13 composition, the restoration time of rheological properties was evaluated at the selected pH. After applying a shear rate of 1000 1 / sec to the sample for 1 minute, the change in storage modulus of 1% IEIK13 was measured at 1 rad / s, 1 Pa using a DHR-1 rheometer (TA Instruments). The IEIK13 composition at the selected pH exhibited typical thixotropic behavior. This means that their rheological properties have been slowly restored. Without wishing to be bound by any particular theory, the restoration time of rheological properties is the time required for the reorganization of peptide molecules to re-form self-association (eg, nanofibers) in the composition. We propose to represent. The complete reorganization time of the 1% IEIK13 control composition (pH 2.3) was up to 12 hours or less, while that of the IEIK13 composition with elevated pH was 6-10 minutes. Representative results for IEIK13 are shown in FIGS. 21A-21D.

Example 4: Rheological Properties of Peptide Compositions as a Function of Ionic Strength This example shows the effect of ionic strength on the rheological properties of a particular peptide composition. In some embodiments, ionic strength can be a control parameter for stiffness, viscosity, and / or restoration time of the peptide composition.

Visual observations of RADA16, KLD12, and IEIK13 compositions containing selected salts (eg KCl, MgCl ₂ , CaCl ₂ ) are summarized in Tables 7-9. The peptide composition at a particular ionic strength was transparent and showed higher stiffness than that at a lower ionic strength. For RADA16 (Table 7), ionic strength in the range of approximately 0.85 to 1.15M (depending on salt identity) did not significantly change the opacity of the RADA16 composition. For KLD12 (Table 8), ionic strength in the range of approximately 0.25 to 0.35M (depending on salt identity) did not significantly change the opacity of the KLD12 composition. For IEIK13 (Table 9), ionic strength in the range of approximately 0.025 to 0.035M (depending on salt identity) did not significantly change the opacity of the IEIK13 composition. The apparent stiffness of the RADA16, KLD12 and IEIK13 compositions increased with increasing ionic strength.

Without wishing to be bound by any particular theory, we propose that the increase in rheological properties may be related to the salting out constant (K) of each salt. The NaCl constant K for RADA16 can be higher than other salts. The rheological properties of the RADA16 composition containing NaCl were slightly higher than those containing KCl and CaCl ₂ .

The critical ionic strength was determined from the visual observations recorded in Tables 7-9. When the ionic strength of the peptide composition was higher than 0.9M (RADA16), 0.3M (KLD12) or 0.03M (IEIK13), the peptide composition began to phase separate. 0.9M, 0.3M and 0.03M may represent critical ionic strengths for RADA16, KDL12 and IEIK13, respectively.

27-29 show rheological properties measured using a rheometer (DHR-1, TA Instruments) equipped with a 20 mm plate when the ionic strength is slightly lower than the critical ionic strength. The ionic strengths of RADA16, KDL12 and IEIK13 were 0.7M, 0.2M and 0.02M for measurement, respectively. The rheological properties of the RADA16, KLD12 and IEIK13 compositions were higher after adjusting their ionic strength levels to 0.7M (RADA16), 0.2M (KLD12) or 0.02M (IEIK13) with NaCl. ..

The rheological properties of the peptide composition at selected ionic strengths were measured using a rheometer (DHR-1, TA Instruments) equipped with a 20 mm plate. The rheological properties of the 1% RADA16 composition were increased by ionic strength regulation up to 0.7M, while decreased at ionic strengths above 0.7M. The rheological properties of the 1% IEIK13 composition were increased by ionic strength regulation up to 0.03M, while decreased at ionic strengths above 0.03M. The results were in good agreement with the visual inspection of the peptide composition at the selected salt ionic strength. The results are shown in FIG. 30 (RADA16) and FIG. 31 (IEIK13).

The viscosity of the peptide composition at the selected ionic strength level was evaluated. The viscosity of the IEIK13 composition increased with increasing ionic strength. The 1% IEIK13 composition exhibited typical shear thinning properties. The viscosity of the 1% IEIK13 composition was evaluated using a rheometer (DHR-1, TA Instruments) equipped with a 20 mm plate. The results are shown in FIGS. 32A and 32B for IEIK13.

After applying high shear stress to the 1% IEIK13 composition at the selected ionic strength, the restoration time of rheological properties was evaluated. After applying a shear rate of 1000 1 / sec to the sample for 1 minute using a DHR-1 rheometer (TA Instruments), the change in storage elastic modulus of 1% IEIK13 was measured by a time sweep test (1rad / s, 1Pa). did. The IEIK13 composition at the selected ionic strength exhibited typical thixotropic behavior and slowly restored their rheological properties. The restoration time of rheological properties is based on the reorganization of peptide molecules that reshape the nanofibers. The complete reorganization time of the 1% IEIK13 control composition without the addition of salt is up to 12 hours or less, while those of the IEIK13 composition containing 0.01M and 0.02M NaCl are 1 It was less than a minute to three minutes. The results for IEIK13 are shown in FIGS. 33A-33C.

Example 5: Rheological properties of the peptide composition as a function of both pH and ionic strength This example shows the rheological properties of the peptide composition at increased pH and ionic strength. Specifically, this example shows the effect of a physiological medium, such as a cell culture medium, on the rheological properties of a particular peptide composition.

The effect of Dulbecco's Modified Eagle's Medium (DMEM) (pH 7.4) on the rheological properties of IEIK13, KLD12, and RADA16 compositions was evaluated using a rheometer equipped with a 40 mm plate (AR500, TA Instruments). DMEM is a cell culture medium containing 6.4 g / L NaCl, 3.4 g / L NaHCO ₃ (sodium bicarbonate), small amounts of other salts, various amino acids, and 4.5 g / L glucose. Is. The pH of DMEM is 7.2 ± 0.2 and the osmotic pressure is 335 ± 30 mOsm / KgH ₂ O. DMEM is close to human physiological fluids (eg blood).

The 1% peptide composition was maintained at 4 ° C. for at least 48 hours prior to mixing with DMEM solution. To carry out the experiment, 1 mL of peptide composition was gently pipetted and placed on a rheometer plate. 2 mL of DMEM solution was gently added around the peptide composition. The peptide composition was treated with DMEM for 2 minutes, then the medium was removed and the plates were placed in a measurement geometric gap near 450 μm. After 2 minutes of relaxation, measurements were taken at 37 ° C. The frequency test was performed at 1 rad / s to 100 rad / s with a vibration stress of 1 Pa.

The rheological properties of the 1% peptide composition were measured before and after 2 minutes of DMEM treatment; the results are shown in FIG. 5A. The rate of increase in storage elastic modulus after DMEM treatment is shown in FIG. 5B. As can be seen, the peptide composition showed a large increase in storage modulus after DMEM treatment. The difference in magnification between RADA16, KLD12, and IEIK13 after DMEM treatment was relatively small compared to that before DMEM treatment. Similarly, the more rigid peptide composition (eg, IEIK13) showed a lower rate of increase in storage modulus after DMEM treatment than the lower rigid peptide composition (eg, RADA16). After DMEM treatment, critical intramolecular interactions increased. It can determine the final stiffness.

Rheological properties of the peptide composition at selected concentrations were measured before and after DMEM treatment using a DHR-1 rheometer (TA Instruments). A frequency sweep test was performed at 1 rad / sec to 10 rad / sec at 1 Pa, and the storage elastic modulus in the graph was 1 rad / sec. The rheological properties of RADA16 and IEIK13 compositions increased with DMEM treatment and / or pH increase. The results are shown in FIGS. 22A and 22B for RADA16 and FIGS. 23A and 23B for IEIK, respectively.

Using DHR-1 rheometers (TA Instruments), the rheological properties of the peptide composition at selected ionic strengths were evaluated 10 minutes after DMEM treatment. The frequency sweep test was performed at 1 rad / sec to 10 rad / sec at 1 Pa, and the storage modulus at 1 rad / sec was selected as the data. The rheological properties of the RADA16 composition were increased by ionic strength regulation up to 0.7M, while they were reduced at 0.7M or higher. At an ionic strength of NaCl of 0.9 M or higher, the RADA16 composition became turbid. The rheological properties of RADA16 were not changed by DMEM treatment (eg, no gelation). However, the rheological properties of the IEIK13 composition were increased by DMEM treatment at the selected ionic strength. The results are shown in FIGS. 34 to 35.

IEIK13, KLD12, and RADA16 were dissolved in salt buffer (eg NaCl) and maintained at elevated pH levels adjusted with alkaline salt buffer (eg NaOH). The compositions had their pH level of about 2.5-4.0 and ionic strength below their critical point. For RADA16, KLD13 and IEIK13, the peptide composition was still transparent at pH 3.4 (adjusted with NaOH) at 0.9% NaCl (ie 0.15M ionic strength). The rheological properties of RADA16 with 0.9% NaCl at pH 3.4 are more rigid than those of RADA16 control (ie, ionic strength and no pH increase) and RADA16 with 0.9% NaCl (no pH increase). rice field. The results are shown in FIG.

As shown in FIG. 1, a Congo red assay was performed to determine gel formation of the peptide composition in PBS (phosphate buffered saline) solution (pH 7.4). 100 μl of each gel at the selected concentration was plated on a glass slide. After 30 seconds, 500 μl of 1% Congo red solution was added around and on top of each of the composition aliquots and then the excess Congo red solution was wiped off prior to the test. RADA16, IEIK13, and KLD12 were plated at selected concentrations of 0.5%, 1.0%, 1.5%, 2.0% and 2.5%. The success or failure of gelation at each concentration was determined by visual observation. RADA16, IEIK13, and KLD gelled at all concentrations.

Example 6: Cell Viability This Example demonstrates the ability of a particular peptide composition to support cell viability. In some embodiments, the peptide compositions provided are characterized in that they support high cell viability (particularly compared to suitable reference compositions).

Cell viability (cytotoxicity) assays were performed to measure the viability of C57 BL / 6 mouse mesenchymal stem cells (mMSC) with the IEIK13, KLD12 and RADA16 compositions described herein. mMSC is a cell line frequently used in hydrogel tissue culture systems. Peptide compositions were prepared at concentrations of 2.5% and then diluted with sucrose to concentrations of 1.5%, 1.25%, 1.0%, 0.75%, and 0.50%. The final concentration of sucrose was 10%. The cells were washed and resuspended in 10% sucrose to a final concentration of 5 million cells / ml. The cells were centrifuged and the supernatant was removed. The cells were resuspended in the peptide composition with 10% sucrose. The protocol then continued plating drop culture and subsequent isolation as described in the PuraMatrix® Usage Guidelines (BD / Corning website). The results for RADA16, IEIK13, and KLD12 are shown in FIGS. 14 to 16, respectively.

Cell viability with the IEIK13 and KLD12 compositions at 0.5% was similar to that at 0.25%. Cell viability with the RADA16 composition at 0.5% is significantly higher than at 0.25%. However, when the peptide concentration exceeded 0.75%, cell viability was significantly reduced. The KLD12 and IEIK13 compositions showed similar or higher cell viability compared to RADA16 at all tested concentrations in the range of 0.25% to 1.5%. The overall cell viability sequence among these peptide compositions was KLD12> IEIK13> RADA16. Tested peptide compositions with concentrations of 0.75% or lower showed cell viability greater than 80%.

Example 7: Rheological Properties of RADA16 Compositions Containing Different Salts This example, among other things, impairs gel reversibility (eg, gel formation and its mechanical integrity after mechanical perturbation), among other things. No), the tests that achieved the adjusted mechanical enhancement of the self-assembled rheological gel are shown. Also, these indicated tests are of gelling kinetics via mixing of cations and anions at selected concentrations in combination with various self-assembling peptides, among other things RADARADARADARADA (or RADA-16). Adjustments have also been achieved.

This example allows the peptide composition to be specifically formulated to have material and / or rheological properties that are particularly useful for a particular application, especially when considered in the context of the present specification. Make sure that the parameters are defined. For example, the techniques described herein may require or benefit from sealants (eg, may require or benefit from increased stiffness), lubricants (eg, may require or benefit from enhanced kinetics). Can receive), drug mixture (eg, may require or benefit from reversible and enhanced kinetics), injectable solution (eg, may require or benefit from reversibility) ) Etc., allowing the preparation of self-assembling peptide compositions specifically adapted to function well. Alternatively or in addition, the techniques described herein can generally assist in the operation and / or production of useful peptide compositions and / or devices containing them. Or allow the selection of parameters contained or applied to them.

For example, reversibility and gel by systematically adjusting the type (eg, Na, K, and Ca) and / or concentration of cations contained in the self-assembled peptide composition, as shown herein. Mechanical strength (ie, stiffness) can be adjusted while still maintaining calcification kinetics. Alternatively or in addition, the gelation kinetics are adjusted while maintaining reversibility by systematically adjusting the type (eg, Cl, SO ₄ , PO ₄ ) and / or concentration of the anion. Can be done.

As shown in this example, in some embodiments (specifically, in embodiments utilizing the RADA16 peptide), Ca was more enhanced in the peptide gel compared to Na and K. Allows rigidity. In addition, in some embodiments (specifically, in embodiments utilizing the RADA16 peptide), Cl allows for faster gelation kinetics in peptide gels as compared to _SO4 . .. In addition, in some embodiments (specifically, in embodiments utilizing the RADA16 peptide), CaCl ₂ optimally mechanically enhances the reversible gel at concentrations of ≧ 0.125 and <0.500M. Allows you to. In the particular test reported in this example, a concentration of ≥0.500M impairs the mechanical properties of the particular gel or makes them unusable for mechanical perturbations after gelation.

In general, the findings reported in this example show that, through the use of various salts and salt concentrations, attributes such as rigidity, gelation kinetics, and gelation reversibility are the peptide concentration, peptide identity (eg, peptide identity). , Amino acid sequence), cation / anion concentration, cation / anion identity, etc. It has been found that both cations and anions can affect attributes independently and in combination. The techniques provided by the present disclosure, including this example, provide a system for adapting a peptide mixture according to desired attributes (eg, performance characteristics) that may be appropriate, for example, for a particular application or situation.

This example specifically shows that certain types of cations and / or anions, and / or their concentrations, have the desired beneficial effect; among others, this example is such. It defines concentrations for anions / cations and the illustrated contexts, and further provides frame arcs that allow one of ordinary skill in the art to do so for other cases (eg, other peptides, etc.).

This example, in particular, and surprisingly, identifies problems with current strategies for providing useful compositions of self-assembling peptides. That is, it is theorized that the ability of self-assembly is dependent on the amount of charged groups available for attack by ionic salts and is the infiltration capacity of the peptide [P. Chen. (2005). "Self-assembly of ionic-complementary peptides: a physicochemical peptide." Colloids and Surfaces]. However, this example demonstrates that, in at least some cases, the peptide is not proportionally affected by salt concentration and therefore mechanical properties and reversibility do not increase linearly and are not rate dependent. do.

41A-41D show protocols used to follow peptide lysis and to assess the effect of specific anions and / or cations and / or their concentrations on a particular RADA16 composition. As shown, the peptide powder in the vial was dissolved in deionized water by vortexing and sonication. The particular peptide composition used in this example is a 1% composition of RADA16, mixed with a 2x salt solution in a 1: 1 ratio to a final concentration of 0.5% RADA16 and a desired molar concentration. I got the salt.

Salt Concentration Test According to the protocol shown in FIGS. 41A-41D, 0.5% solutions of RADA16 containing different concentrations of CaCl ₂ were prepared. The mixed solution was allowed to sit for a relaxation period of approximately 24 hours. The vial was then turned upside down so that the gel properties could be evaluated. If the composition remained perfectly in place when the vial containing it was turned upside down, the composition was considered to be a fully functional gel. A semi-functional gel was considered if more than half of the composition remained in place when the vial containing it was turned upside down. Upon inversion, more than half of the composition, and especially if substantially all of the composition fell to the opposite end of the vial containing it, was considered a non-functional gel.

42A-42G show photographs of the results obtained, specifically upright and inverted vials relating to CaCl ₂ at various concentrations, respectively. As can be seen, for these 0.5% RADA16 compositions, 0.250M CaCl ₂ forms a fully functional gel (panel E) and 0.500M CaCl ₂ forms a semi-functional gel. (Panel F) and 1M CaCl ₂ formed a non-functional gel (Panel G).

Cation Selection Test 0.5 of RADA16 containing 0.005, 0.05, 0.125, 0.25, 0.5, and 1M NaCl, KCl, and CaCl ₂ according to the protocol shown in FIGS. 41A-41D. % Solution was prepared. The effects of cations, sodium (Na ⁺ ), potassium (K ⁺ ), and calcium (Ca ²⁺ ) were observed, keeping the anions and chlorides (Cl ⁻ ) identical. The results are shown in FIG. 43 to provide a basic understanding of how cation changes in salt solutions affect the viscoelastic properties and stiffness of the resulting composition.

Mechanical Strength Test According to the protocol shown in FIGS. 41A-41D, the stiffness of the peptide composition containing either 2.5% RADA16 and no salt, or 2.5% RADA16 and 0.125M CaCl ₂ . analyzed. The results are shown in FIG. 44 to provide a basic understanding of the viscous properties of the resulting composition. As can be seen, there is a significant increase in stiffness between the two solutions when the cationic solutions are mixed.

Reversibility Test A solution of 0.5% RADA16 mixed with 0.125, 0.25, or 0.5M CaCl ₂ was prepared according to the protocol shown in FIGS. 41A-41D. The compositions were subjected to mechanical stresses through vortexing and sonication so that their structures were completely destroyed (eg, randomized). The destroyed composition was then left at room temperature for 48 hours to allow self-organization to occur. FIGS. 45A and 45B show the results of this test, revealing the basic viscoelastic properties of the peptide composition and, if containing 0.125 or 0.25 M CaCl ₂ , perturbation of any structure in the composition. It is shown that reversibility can be maintained even later. In contrast, compositions containing 0.5 M CaCl ₂ show dramatically lower stiffness after disintegration (reflecting the dramatically reduced ability to restore structure).

Equivalents One of ordinary skill in the art will be able to recognize or confirm many equivalents for a particular embodiment of the invention described herein using routine experiments at best. The scope of the invention is not intended to be limited to the above description, but rather is set forth in the following claims.
[References]
[1] P. Chen. (2005). “Self-assembly of ionic-complementary peptides: a physicochemical viewpoint.” Colloids and Surfaces .
[2] Mishra A., et al. (2013). “Influence of metal salts on the hydrogelation properties of ultrashort polypeptides peptides.” RSC Advances .
[3] Shuguang, Z., et al. (1999). “Peptide self-assembly in functional polymer science and
Engineering. ” Reactive and Functional Polymers .
[4] Yanlian, Y., et al. (2009). “Designer self-assembling peptide nanomaterials.” Nano Today .
[5] Zhaoyang, Y., et al. (2008). “Temperature and pH effects on biophysical and morphological properties of self-assembling peptide RADA16-I.” Journal of Peptide Science .

Claims (20)

It is an IEIK13 composition and
Contains the IEIK13 peptide at a concentration of at least 0.25%;
The composition has a pH in the range of about 2.5 to about 4.0.
IEIK13 composition. The composition of claim 1.
The composition is a solution,
Composition. The composition of claim 1.
The composition is a gel,
Composition. The composition of claim 1.
The composition has an ionic strength in the range of about 0.0001M to about 0.1M.
Composition. The composition of claim 4.
The ionic strength is regulated / imparted by common salts.
The general salts are selected from the group consisting of NaCl, KCl, MgCl ₂ , CaCl ₂ and CaSO ₄ .
Composition. The composition of claim 4.
The ionic strength is imparted by common salts, and the ionic strength is imparted.
The common salts consist of one or more salts forming cations and one or more salts forming anions.
The salt forming the cation is selected from the group consisting of ammonium, calcium, iron, magnesium, potassium, pyridinium, quaternary ammonium, and sodium.
The salt forming the anion is selected from the group consisting of acetate, carbonate, chloride, citrate, cyanide, flolide, nitrate, nitrite, and phosphate.
Composition. The composition of claim 1.
The composition has a storage elastic modulus in the range of about 100 to about 10,000 Pa at a frequency of 1 rad / sec and a vibration stress of 1 Pa.
Composition. The composition of claim 1.
The composition is buffered with sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, sodium acetate, sodium sulfide, or DMEM.
Composition. The composition of claim 1.
The IEIK13 peptide is present at a concentration of less than 3% and / or
The composition has a pH in the range of about 3.0 to about 4.0.
Composition. The composition of claim 9.
The composition is a gel exhibiting a storage modulus of more than 500 Pa at a frequency of 1 rad / sec and a vibration stress of 1 Pa.
Composition. The composition of claim 1.
The composition is buffered with sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, sodium acetate, or sodium sulfide so that the pH of the composition is regulated.
Composition. A liquid peptide composition
Contains peptides having amino acid lengths in the range of about 6 to about 20 and alternating amino acid sequences of hydrophobic and hydrophilic amino acids.
The composition is characterized by the following:
It has a viscosity in the range of about 1 Pa · s to about 500,000 Pa · s at room temperature.
With a frequency of 1 rad / sec and a vibration stress of 1 Pa, it has a storage elastic modulus in the range of about 1 to about 5000 Pa.
Within a time of about 0 to about 30 s when exposed / maintained to a pH in the range of about 2.5 to about 4.0 and / or an ionic strength in the range of about 0.0001 M to about 1.5 M. , Forming a gel,
Liquid peptide composition. The composition of claim 12.
Aqueous composition,
Composition. The composition of claim 12.
The peptide comprises RADA16.
Composition. The composition of claim 12.
The peptide comprises IEIK13.
Composition. The composition of claim 12.
The peptide comprises KLD12.
Composition. The composition of claim 12.
The ionic strength is imparted by common salts, and the ionic strength is imparted.
The general salts are selected from the group consisting of NaCl, KCl, MgCl ₂ , CaCl ₂ and CaSO ₄ .
Composition. The composition of claim 12.
The ionic strength is imparted by common salts, and the ionic strength is imparted.
The common salts consist of one or more salts forming cations and one or more salts forming anions.
The salt forming the cation is selected from the group consisting of ammonium, calcium, iron, magnesium, potassium, pyridinium, quaternary ammonium, and sodium.
The salt forming the anion is selected from the group consisting of acetate, carbonate, chloride, citrate, cyanide, flolide, nitrate, nitrite, and phosphate.
Composition. The composition of claim 12.
The composition is buffered with sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, sodium acetate, or sodium sulfide so that the pH of the composition is regulated.
Composition. A method comprising selecting a peptide composition for application to a particular in vivo site.
The method is
A step of determining one or more parameters selected from the group consisting of storage modulus, viscosity, gelling time, shear de-viscosity properties, and peptide nanofiber reassembly time for the peptide composition; and said. The step of comparing one or more of the determined parameters of the above with a set of properties determined to be suitable for application to the particular in vivo site; and selecting the peptide composition in light of the comparison. Steps; and the step of administering the selected peptide composition to the site,
including,
Method.

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